(Not applicable)
This invention relates to the field of adenoviral vectors and transfection. More specifically, the invention relates to target cell-specific adenoviral vectors containing E3.
In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Neoplasia resulting in benign tumors can usually be completely cured by removing the mass surgically. If a tumor becomes malignant, as manifested by invasion of surrounding tissue, it becomes much more difficult to eradicate. Once a malignant tumor metastasizes, it is much less likely to be eradicated.
A major, indeed the overwhelming, obstacle to cancer therapy is the problem of selectivity; that is, the ability to inhibit the multiplication of tumor cells, while leaving unaffected the function of normal cells. For example, in prostate cancer therapy, the therapeutic ratio, or ratio of tumor cell killing to normal cell killing of traditional tumor chemotherapy, is only 1.5:1. Thus, more effective treatment methods and pharmaceutical compositions for therapy and prophylaxis of neoplasia are needed.
One possible treatment approach for many of these cancers is gene therapy, whereby a gene of interest is introduced into the malignant cell. Various viral vectors, including adenoviral vectors, have been developed as vehicles for gene therapy. The virtually exclusive focus in development of adenoviral vectors for gene therapy is use of adenovirus merely as a vehicle for introducing the gene of interest, not as an effector in itself. Replication of adenovirus has been viewed as an undesirable result. In the treatment of cancer by replication-defective adenoviruses, the host immune response limits the duration of repeat doses at two levels. First, the capsid proteins of the adenovirus delivery vehicle itself are immunogenic. Second, viral late genes are frequently expressed in transduced cells, eliciting cellular immunity to the virus-infected cells. Thus, the ability to repeatedly administer cytokines, tumor suppressor genes, ribozymes, suicide genes, or genes which convert prodrug to an active drug has been limited by the immunogenicity of both the gene transfer vehicle and the viral gene products of the transfer vehicle as well as the transient nature of gene expression.
Adenovirus can cause persistent infections in humans and animals. The strategies of C type adenovirus (type Ad2 and Ad5) for evading host immune recognition are many, and generally involve E3, a delayed early transcription unit whose transcription is induced by the 289R E1A protein. During early stages of infection, the E3 promoter drives expression of nine alternatively spliced mRNAs that are polyadenylated at one of two sites, E3A and E3B. Wold et al. (1995) Curr. Topics Microbiol. Immunol199 (Pt.1):237-274. None of the E3 proteins is apparently required for adenovirus replication in cultured cells or in the lungs of hamsters or cotton rats, but they appear to play a role in evasion of host immune surveillance.
Six proteins which are encoded by the Ad-E3 region have been identified and characterized: (1) a 19-kDa glycoprotein (gp19k) is one of the most abundant adenovirus early proteins, and is known to inhibit transport of the major histocompatibility complex class I molecules to the cell surface, thus impairing both peptide recognition and clearance of Ad-infected cells by cytotoxic T lymphocytes (CTLs); (2) E3 14.7 k protein and the E3 10.4 k/14.5 k complex of proteins inhibit the cytotoxic and inflammatory responses mediated by tumor necrosis factor (TNF); (3) E3 10.4 k/14.5 k protein complex downregulates the epidermal growth factor receptor, which may inhibit inflammation and activate quiescent infected cells for efficient virus replication; (4) E3 11.6 k protein (adenoviral death protein, ADP) from adenovirus 2 and 5 appears to promote cell death and release of virus from infected cells. Other studies have indicated that the E3-encoded 10.4K/14.5K complex proteins down-modulate the apoptosis receptor Fas/Apo-1. Elsing and Burger (1998) Proc. Natl. Acad. Sci. USA 95:10072-10077; and Shisler et al. (1997) J. Virol. 71:8299-8306. The functions of three E3-encoded proteinsxe2x80x943.6 k, 6.7 k, and 12.5 kxe2x80x94are currently unknown. Wold et al. (1995).
Traditionally, the pervasive dogma regarding the role of E3 in adenoviral vectors for gene therapy was that E3 should be deleted. E3 was viewed as non-essential for replication, and its deletion allowed insertion of foreign genes. Indeed, until quite recently, all adenoviral vectors lacked the E3 region.
More recently, it has been demonstrated that incorporation of E3 genes in the engineered adenovirus reduces the antiviral immune response and prolongs expression of foreign genes delivered by adenoviral vectors. It was shown that insertion of E3 genes in recombinant adenovirus facilitates re-administration of a functional vector for long-term gene expression and correction of an inherited metabolic disorder. Horwitz et al. (1995) Curr. Topics Microbiol. Immunol. 199(Pt 1):195-211. Other studies have indicated that, while expression from E3-deleted vectors is essentially turned off eight weeks after gene transfer, an E3-containing vector supported transgene expression with therapeutic levels of human factor IX in vivo for more than 4 months. Poller et al. (1996) Gene Ther. 3:521-530. The enhanced stability was attributed to efficient E3 region-mediated suppression of the host""s antiviral immune response. More recently, it was demonstrated that a wild-type E3-containing adenoviral vector could direct prolonged expression of a non-immunogenic transgene. Persistence of this gene expression was also attributed to the presence of the B3 region. Wadsworth et al. (1997) J. Virol. 71:5189-5196. Further, when a recombinant adenovirus vector encoding hepatitis B surface antigen and containing an intact E3 region was used to infect chimpanzees, greater viral persistence, as indicated by the duration of virus shedding, was observed compared to counterpart vectors lacking E3. This phenomenon was attributed to evasion of host immune response. Chengalvala et al. (1997) Vaccine 15:335-339. However, the above-described E3-containing adenoviral vectors were not replication competent and target cell specific. All of these studies employed adenovirus as a vehicle for expressing a transgene.
Use of adenoviral vectors as therapeutic vehicles for cancer has been reported. See, for example, Bischoff et al. (1996) Science 274:373-376; WO 96/349969; WO 96/17053. Some of these approaches utilize target cell-type specific transcriptional regulatory elements to selectively drive adenoviral replication (and thus cytotoxicity). U.S. Pat. No. 5,698,443; see also WO 95/11984; WO 96/17053; WO 98/39465; WO 98/39467; WO 98/39466; and WO 98/39464. These vectors were deleted for E3.
Based on the teachings of the prior art, inclusion of E3 is not indicated in the context of using adenoviral vector replication for its cytotoxic effects (as opposed to using an adenoviral vectors as gene delivery vehicles), as suppression of the host""s cytotoxic T cell response would not be considered a positive or desirable result. Further, inclusion of E3 into replication-competent adenoviral vectors would not be indicated since the well-accepted understanding in the art is that E3 is not necessary for viral replication.
Besides cancerous cells, it is often desirable to selectively destroy certain unwanted cells or tissues. Apart from surgery, however, which is invasive, there is a dearth of methods available, particularly non-invasive methods, which would allow such selective cytotoxicity and/or suppression.
There is a need for vector constructs that are capable of rapidly eliminating cancerous cells in a minimum number of administrations and which are suitable for use in cancer ablation treatments. There is also a need for an ability to selectively destroy, or impair, unwanted cells, regardless of cell type and/or regardless of anatomical location.
All publications and patent applications cited herein are hereby incorporated by reference in their entirety.
The invention provides target cell-specific adenoviral vectors (i.e., they are preferentially cytotoxic toward a specific target cell) comprising E3 (or an E3 sequence, or a portion of an E3 region), compositions, host cells, and kits comprising these vectors, and methods using these vectors. Preferably, the vectors are replication competent. These target cell-specific vectors express E3-encoded protein(s) and exhibit significantly greater cytotoxicity and/or enhanced adenoviral production per cell.
Accordingly, in one aspect, the invention provides an adenovirus vector comprising (a) an adenovirus gene under transcriptional control of a target cell-specific transcriptional regulatory element (TRE); and (b) an E3 region. In another aspect, an adenovirus vector of the invention comprises (a) an adenovirus gene under transcriptional control of a target cell-specific transcriptional regulatory element (TRE); and (b) an E3 sequence. In some embodiments the adenoviral gene under transcriptional control of a target cell-specific TRE is one that is essential for adenoviral propagation, such as E1A and/or E1B. In some embodiments, the target cell-specific TRE confers cell type-specific transcriptional regulation on the operably linked adenoviral gene. In other embodiments, the target cell-specific TRE is a cell status-specific TRE.
In other embodiments, the invention provides an adenovirus vector comprising (a) an adenovirus gene under transcriptional control of a target cell-specific transcriptional regulatory element (TRE); and (b) a portion of an E3 region.
In another aspect, the invention provides a replication competent adenovirus vector comprising an E3 sequence (or E3 region) under transcriptional control of a target cell specific TRE.
In another aspect, E3-containing, target-cell-specific adenoviral vectors of the invention further contain one or more transgenes. In some of these embodiments, a transgene can be under transcriptional control of a heterologous TRE, which may be a target cell-specific TRE.
In another aspect, the invention provides an adenoviral vector comprising an E3 region (or a portion of an E3 region, or an E3 sequence) under transcriptional control of a target-cell specific TRE.
In another aspect, the invention provides a host cell comprising an adenovirus vector(s) described herein.
In another aspect, the invention provides compositions comprising an adenovirus vector(s) described herein, preferably further comprising a pharmaceutically acceptable excipient.
In another aspect, the invention provides kits which contain an adenoviral vector(s) described herein.
In another aspect, methods are provided for propagating an adenovirus specific for mammalian cells which permit the function of a target cell-specific TRE, said method comprising combining an adenovirus vector(s) described herein with mammalian cells that permit the function of a target cell-specific TRE, such that the adenovirus vector(s) enters the cell, whereby said adenovirus is propagated.
In another aspect, methods are provided for conferring selective cytotoxicity in target cells, comprising contacting the cells with an adenovirus vector(s) described herein, whereby the vector enters the cell.
The invention further provides methods of suppressing tumor cell growth, more particularly a target tumor cell, comprising contacting a tumor cell with an adenoviral vector of the invention such that the adenoviral vector enters the tumor cell and exhibits selective cytotoxicity for the tumor cell.
In another aspect, methods are provided for detecting a cell which allows the function of a target cell-specific TRE, which comprise contacting a cell in a biological sample with an adenovirus vector(s) of the invention, and detecting replication of the adenovirus vector(s), if any.